Heretofore, technology of crystallography is indispensable in order to elucidate a function of a biopolymer represented by protein. The crystal structure of a biopolymer is analyzed by using nuclear magnetic resonance (NMR) measurement, X diffraction measurement, neutron diffraction measurement, etc. By using data of analyzed crystal structure, further analysis for higher-order structure and function of the biopolymer can be carried out.
For such crystallography and evaluation of a biopolymer, it is required to produce a good crystal.
In recent years, the number of a biopolymer having known a crystallization condition is increased rapidly. However, a systematic theory about formation of a crystalline nucleus and crystal growth is not found.
Here, as a factor for crystallizing a biopolymer, various kinds factor are considered: for example, solution concentration of a biopolymer, a type of a precipitant and its concentration, a type of buffer solution and its pH value, temperature of solution, etc. Formerly, for each biopolymer, it is experimented by trial and error about the factor of such crystallization, respectively. Thus, search of crystallization conditions was inefficient.
In addition, the crystal growth rate of the biopolymer in solution was very slow, formerly. Therefore, even under most suitable crystallization condition, for being able to confirm formation of a crystal with an optical microscope etc., time from at least several days to several weeks, or for several months or more, depending on a case, was required.
In this way, production of the biopolymer crystal had required great time and effort.
In addition, in order to have made the good biopolymer crystal suitable for crystallography, high-purity biopolymer materials were used.
Therefore, by a cost problem, it was preferred that the amount of solution used by crystal production is very small quantities, for example, less than several tens of micro L.
However, when a crystal is prepared by comprehensive searching for crystallization conditions inefficiently, volume more than that was needed.
Therefore, a promoting method of crystallization of a biopolymer, which does not depend on search of inefficient and comprehensive crystallization conditions on the above, is desired.
Formerly, a photoirradiation method, the strong magnetic field applying method, the electric field applying method, etc., are proposed as a promoting method of such crystallization.
Especially in these, crystallization by photoirradiation or electric field application was easy practicing method, comparatively.
Firstly, as refer to the patent documents 1, it is disclosed about the manufacturing method of biopolymer crystals, such as protein by the conventional photoirradiation, especially ultraviolet rays, and its manufacturing device. In the crystallization method by the conventional photoirradiation, which was described in the patent documents 1, in many cases, the ultraviolet rays having wavelength about 200-350 nm in which the optical absorption of a biopolymer occurs were used.
However, irradiation of energy-rich ultraviolet rays changed molecular structure of a biopolymer itself and had the problem to damage to the biopolymer directly. Thus, a crystallization method by photoirradiation was not preferred in order to obtain a good crystal of a biopolymer.
In a crystallization method by the conventional electric field application on the other hand as roughly classified, two kinds of methods, the internal electric field applying method and the external electric field applying method, mainly existed.
The internal electric field applying method is a crystallization method which contacts electrodes of voltage application in crystallization solution directly, and applies an electric field. The external electric field applying method is a crystallization method which puts an insulator near electrodes and applies an electric field to crystallization solution indirectly.
A crystallization method by electric field application, such as these internal electric field applying method and the external electric field applying method, unlike ultraviolet irradiation, little damage to a biopolymer is observed if a low electric field is applied.
Here, the internal electric field applying method is the voltage application method to contact electrodes in crystallization solution directly, as mentioned above.
Formerly, in the internal electric field applying method, conductive metals, such as copper, silver, gold, and platinum, were used as an electrode for applying an electric field to crystallization solution.
However, these metallic materials had a problem that a surface of metal generally served as a crystalline nucleus of a biopolymer in many cases.
Hence, there was a case where a lot of crystallites were formed near a metal electrode at once regardless of existence of electric field application to crystallization solution. By this reason, it might become the disturbance of good crystal formation.
Therefore, the conventional internal electric field applying method had a problem that control of crystal growth was difficult.
Thus, as a crystallization method by electric field application, the external electric field applying method is frequently used from the former.
In such conventional external electric field applying methods, it is devised in order that the electrode itself may not become the impediment of crystal formation by insertion of an insulating material near a metal electrode, or insulating layer formation on a metal electrode.
For example, as refer to nonpatent document 1, a test method of protein crystal growth by the conventional external electric field applying method is described.
Also, as refer to nonpatent document 2, for example, a crystallizing device of protein by the conventional external electric field applying method is reported.
In technology of the nonpatent document 1 and the nonpatent document 2, a high voltage of a direct current from several hundreds V to several tens of kV is applied to inter-electrode of the metal electrodes covered with an insulating material, respectively. Thereby, it performs to crystallize a biopolymer easier.
Furthermore, in the patent documents 2, as a kind of the conventional external electric field applying method, a apparatus which applies and crystallize by applying alternating voltage to protein crystallization solution in a micro fluid device is disclosed.
The technology of the patent documents 2 is the method of performing crystal formation according to dielectrophoresis force by an electric field acted in protein and carrying the crystal. Also in this technology, high alternating voltage needed to be applied to inter-electrode on account of configuration of electrodes in the micro fluid.